PROJECT SUMMARY Chromatin function is regulated by reversible histone post-translational modifications (PTMs), including lysine methylation, acetylation, and monoubiquitination. Evidence places histone monoubiquitination (ub1) upstream of other chromatin PTMs, acting as a critical signaling center that regulates cascades of downstream epigenetic enzymes (e.g. histone methyltransferase enzymes or HMTs) to modify gene transcription. Indeed, chromatin-targeting deubiquitination enzymes (i.e. DUBs) are emerging as attractive therapeutic targets as these enzymes are associated with disease and potentially highly druggable. However, current high-throughput screening (HTS) DUB assays typically use unnatural ubiquitin substrate analogs (e.g. Ub-AMC or diubiquitin conjugates), which fail to model the target-specific interactions observed in vivo. This is significant as the activities of several chromatin-targeting DUBs are dramatically increased when targeting nucleosome substrates (the basic repeating unit of chromatin) vs. modified peptides or unnatural ubiquitin analogs. Here, EpiCypher will develop first-to-market HTS assays that utilize ?ubiquitinated designer nucleosomes? (Ub-dNucs) as biochemical substrates. The innovation of this project is the commercial manufacturing of Ub-dNucs together with the use of these reagents to establish chromatin-dependent DUB / HMT assays for precision therapeutic development. In Phase II, we will generate a set of disease-relevant Ub-dNucs as well as scale-up commercial manufacturing to support HTS assay development (Aim 1). Using these Ub-dNucs as substrates, we will develop the first commercial chromatin-based HTS DUB assays on the market (Aim 2), providing access to validated DUB targets that remain challenging to study using non-physiological substrates (e.g. ubiquitin analogs currently used for HTS). In addition to DUB assays, we will also establish a novel approach to identify target-specific inhibitors for HMTs. This innovative assay strategy capitalizes on the dramatic stimulatory effect that histone ub1 has on the activity of downstream epigenetic enzymes, many of which are high value drug targets. Finally, we will perform a series of pilot compound screens using selected Ub-dNuc-dependent DUB and HMT assays (Aim 3). The goal of these studies is to demonstrate how Ub-dNucs can be used to identify true, validated hits that would otherwise be missed using current substrates (e.g. Ub analogs for DUBs and unmodified nucleosomes / peptide for HMTs). The breakthrough technology developed herein will provide access to high-value DUB targets, which are inaccessible using current biochemical approaches, and enable development of precision therapeutics for Ub-dependent HMTs.